Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent epiblast cells by orchestrated exposures to cytokines, including bone morphogenetic protein 4 (Bmp4). In human embryos, the earliest PGCs have been identified on the endodermal wall of yolk sac around the end of the 3rd week of gestation, but little is known about the process of human PGC specification and their early development. To circumvent the technical and ethical barriers of studying human embryonic PGCs, surrogate cell culture models have been recently generated from pluripotent stem cells. Here, we describe a 13-day protocol for robust production of human PGC-Like Cells (hPGCLCs). Human induced pluripotent stem cells (hiPSCs) maintained in the primed pluripotency state are incubated in the 4i naïve reprogramming medium for 48 hours, dissociated to single cells, and packed into microwells. Prolonged maintenance of hiPSCs in the naïve pluripotency state causes significant chromosomal aberrations and should be avoided. hiPSCs in the microwells are maintained for an additional 24 hours in the 4i medium to form embryoid bodies (EBs), which are then cultured in low-adherence plasticware under a rocking condition in the hPGCLC induction medium containing a high concentration of recombinant human BMP4. EBs are further cultured for up to 8 days in the rocking, non-adherent condition to obtain maximum yields of hPGCLCs. By immunohistochemistry, hPGCLCs are readily detected as cells strongly expressing OCT4 in almost all EBs exclusively on their surface. When EBs are enzymatically dissociated and subjected to FACS enrichment, hPGCLCs can be collected as CD38+ cells with up to 40-45% yield.

Abstract A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-L1 expression and mutational burden, are insufficiently precise to guide treatment selection and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma Circulating Tumor Cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC-Score) applied to microfluidically-enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (N=42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC-Score within 7 weeks of therapy correlates with marked improvement in progression-free survival (Hazard Ratio (HR): 0.17, P=0.008) and overall survival (HR: 0.12, P=0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies. Citation Format: Xin Hong, Ryan J. Sullivan, Mark Kalinich, Tanya Kwan, Shiwei Pan, Joseph A. LiCausi, John D. Milner, Linda T. Nieman, Ben S. Wittner, Uyen Ho, Tianqi Chen, Ravi Kapur, Don Lawrence, Keith T. Flaherty, Lecia V. Sequist, Sridhar Ramaswamy, David T. Miyamoto, Michael Lawrence, Anita Giobbie-Hurder, Mehmet Toner, Kurt J. Isselbacher, Shyamala Maheswaran, Daniel A. Haber. Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-144.

Significance Identifying predictive biomarkers of therapeutic response for melanoma patients treated with immune checkpoint inhibitors is a major challenge. By combining microfluidic enrichment for melanoma circulating tumor cells (CTCs) together with RNA-based droplet digital PCR quantitation, we have established a highly sensitive and robust platform for noninvasive, blood-based monitoring of tumor burden. Serial monitoring of melanoma patients treated with immune checkpoint inhibitors shows rapid changes in CTC score, which precede standard clinical assessment and are highly predictive of long-term clinical outcome. Early on-treatment digital monitoring of CTC dynamics may thus help identify patients likely to benefit from immune checkpoint inhibition therapy.

Abstract Background: Although liver cancer has the second-highest mortality rate among cancers internationally, accurate and scalable assays for the early detection and longitudinal monitoring of hepatocellular carcinoma are lacking. Circulating tumor cells are released from invasive cancers into the blood stream, but the difficulty inherent in isolating, identifying, and characterizing these ultra-rare cells has precluded their widespread implementation as a biomarker. By combining a high-throughput microfluidic negative depletion CTC isolation strategy with the absolute quantification of lineage-specific RNAs, we report the highly specific detection of hepatocellular carcinoma CTCs from patient blood draws. Methods: Blood draws from 48 hepatocellular carcinoma patients, 31 chronic liver disease patients, 25 healthy donors, and 44 patients with primary cancers other than hepatocellular carcinoma were processed through the microfluidic device (CTC-iChip). RNA was extracted, whole-transcriptome amplified, and quantified using droplet digital PCR. Transcript counts were used to fit a logistic regression model to integrate distinct transcript levels into a single CTC-score. The technical feasibility of utilizing RNA sequencing for identification of novel CTC transcripts of interest was also demonstrated with a liver cancer cell line spike-in study. Results: 9 of the 16 untreated HCC patients were successfully detected, while only 1/31 chronic liver disease patients were incorrectly classified. HCC patients undergoing treatment showed a significant decrease in their CTC-score; only 9/32 patients actively receiving treatment were positive. The CTC-score was not correlated with the HCC serum biomarker alpha-fetoprotein, and combining these two orthogonal measures led to estimated positive and negative predictive values of 80% and 86%, respectively, in a high-risk cohort. RNAseq analysis of cell line spike-in data revealed the potential of RNA sequencing for uncovering novel transcripts of interest. Conclusion: Coupling microfluidic depletion with droplet digital PCR allows for the highly specific detection of hepatocellular carcinoma. The CTC-score generated from these data tracks with clinical intervention and is orthogonal to the existing biomarker AFP; combining these two assays has the potential to provide superior detection compared to either individual approach. Citation Format: Mark Kalinich, Irun Bhan, Tanya T. Kwan, David T. Miyamoto, Sarah Javaid, Joseph A. LiCausi, John D. Milner, Xin Hong, Lipika Goyal, Srinjoy Sil, Melissa Choz, Ravi Kapur, Alona Muzikansky, Huidan Zhang, David A. Weitz, Lecia V. Sequist, David P. Ryan, Raymond Chung, Andrew X. Zhu, Kurt J. Isselbacher, David T. Ting, Mehmet Toner, Shyamala Maheswaran, Daniel A. Haber. Absolute quantification of circulating tumor cell RNA enables high specificity detection of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1734. doi:10.1158/1538-7445.AM2017-1734

Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer. We recently described a high-throughput microfluidic CTC-iChip, which efficiently depletes hematopoietic cells from blood specimens and enriches for CTCs with well-preserved RNA. Application of RNA-based digital PCR to detect CTC-derived signatures may thus enable highly accurate tissue lineage-based cancer detection in blood specimens. As proof of principle, we examined hepatocellular carcinoma (HCC), a cancer that is derived from liver cells bearing a unique gene expression profile. After identifying a digital signature of 10 liver-specific transcripts, we used a cross-validated logistic regression model to identify the presence of HCC-derived CTCs in nine of 16 (56%) untreated patients with HCC versus one of 31 (3%) patients with nonmalignant liver disease at risk for developing HCC (P < 0.0001). Positive CTC scores declined in treated patients: Nine of 32 (28%) patients receiving therapy and only one of 15 (7%) patients who had undergone curative-intent ablation, surgery, or liver transplantation were positive. RNA-based digital CTC scoring was not correlated with the standard HCC serum protein marker alpha fetoprotein (P = 0.57). Modeling the sequential use of these two orthogonal markers for liver cancer screening in patients with high-risk cirrhosis generates positive and negative predictive values of 80% and 86%, respectively. Thus, digital RNA quantitation constitutes a sensitive and specific CTC readout, enabling high-throughput clinical applications, such as noninvasive screening for HCC in populations where viral hepatitis and cirrhosis are prevalent.

Pluripotent stem cell-derived human primordial germ cell-like cells (hPGCLCs) provide important opportunities to study primordial germ cells (PGCs). We robustly produced CD38+ hPGCLCs [∼43% of FACS-sorted embryoid body (EB) cells] from primed-state induced pluripotent stem cells (iPSCs) after a 72-hour transient incubation in the four chemical inhibitors (4i)-naïve reprogramming medium and showed transcriptional consistency of our hPGCLCs with hPGCLCs generated in previous studies using various and distinct protocols. Both CD38+ hPGCLCs and CD38- EB cells significantly expressed PRDM1 and TFAP2C, although PRDM1 mRNA in CD38- cells lacked the 3'-UTR harboring miRNA binding sites regulating mRNA stability. Genes up-regulated in hPGCLCs were enriched for cell migration genes, and their promoters were enriched for the binding motifs of TFAP2 (which was identified in promoters of T, NANOS3, and SOX17) and the RREB-1 cell adhesion regulator. In EBs, hPGCLCs were identified exclusively in the outermost surface monolayer as dispersed cells or cell aggregates with strong and specific expression of POU5F1/OCT4 protein. Time-lapse live cell imaging revealed active migration of hPGCLCs on Matrigel. Whereas all hPGCLCs strongly expressed the CXCR4 chemotaxis receptor, its ligand CXCL12/SDF1 was not significantly expressed in the whole EBs. Exposure of hPGCLCs to CXCL12/SDF1 induced cell migration genes and antiapoptosis genes. Thus, our study shows that transcriptionally consistent hPGCLCs can be readily produced from hiPSCs after transition of their pluripotency from the primed state using various methods and that hPGCLCs resemble the early-stage PGCs randomly migrating in the midline region of human embryos before initiation of the CXCL12/SDF1-guided chemotaxis.

Dr. Haber: Circulating tumor cells are cancer cells that are found in the blood. This is how cancer spreads from a primary tumor in an organ to give rise to metastases. These cells are rare, and they’re surrounded by many blood cells. We’ve spent a lot of time developing technologies to pull these cells out of the blood so we can study both metastasis, as well as cancer progression. Some techniques use the fact that cancer cells are somewhat larger than blood cells so they try to filter them, but some cancer cells are not that large so you can miss a lot that way. Other technologies involve using antibodies against epitopes on tumor cells or epithelial cells to pull them out; those have become standard in the field. The challenge is that some epithelial cells become more mesenchymal and some cancer cells have different epitopes, so it’s hard to get a reliable antibody to always pull out tumor cells. The technologies that we’ve used in collaboration with Mehmet Toner, a bioengineer at Massachusetts General Hospital, purify out the normal cells. If you use an antibody against white blood cells to deplete them from a blood specimen and you remove the red blood cells and platelets, then you’re left with the cells that don’t belong in the blood: the tumor cells. That’s hard to do; you need a technology that is really efficient at purifying away a large excess of normal blood cells. That’s a big advantage in the field of microfluidics, where you can line up the cells in such a way that they pass single file through a channel. You can attach a magnetic particle with an antibody to the cells of interest and then pass them through a magnet so you can separate them. The technology has gotten more complex but also more powerful, to the point where you can reliably pull out these cells and ask the questions that you’ve always wanted to ask. Elizabeth McKenna: Are CTCs similar to the parental tumor or have they diverged in any way? How are they generated and which ones are selected for metastasizing?

Although biological effects of endocrine disrupting chemicals (EDCs) are often observed at unexpectedly low doses with occasional nonmonotonic dose-response characteristics, transcriptome-wide profiles of sensitivities or dose-dependent behaviors of the EDC responsive genes have remained unexplored. Here, we describe expressome analysis for the comprehensive examination of dose-dependent gene responses and its applications to characterize estrogen responsive genes in MCF-7 cells. Transcriptomes of MCF-7 cells exposed to varying concentrations of representative natural and xenobiotic estrogens for 48 h were determined by microarray and used for computational calculation of interpolated approximations of estimated transcriptomes for 300 doses uniformly distributed in log space for each chemical. The entire collection of these estimated transcriptomes, designated as the expressome, has provided unique opportunities to profile chemical-specific distributions of ligand sensitivities for large numbers of estrogen responsive genes, revealing that at low concentrations estrogens generally tended to suppress rather than to activate transcription. Gene ontology analysis demonstrated distinct functional enrichment between high-and low-sensitivity estrogen responsive genes, supporting the notion that a single EDC chemical can cause qualitatively distinct biological responses at different doses. Expressomal heatmap visualization of dose-dependent induction of Bisphenol A inducible genes showed a weak gene activation peak at a very low concentration range (ca. 0.1 nM) in addition to the main, strong gene activation peak at and above 100 nM. Thus, expressome analysis is a powerful approach to understanding the EDC dose-dependent dynamic changes in gene expression at the transcriptomal level, providing important information on the overall profiles of ligand sensitivities and nonmonotonic responses.

Niacin The mechanism of action of niacin is not fully understood, but it appears to inhibit the secretion of lipoproteins containing apo B100 from the liver. Niacin decreases both total and LDL cholesterol approximately 15 to 25%, reduces VLDL levels by 25 to 35%, and raises HDL cholesterol levels by as much as 15 to 25%. Thus, niacin exerts favorable changes on the three major lipoproteins (VLDL, LDL, and HDL). Efficacy of monotherapy was confirmed in a longterm secondary prevention trial in which niacin significantly reduced the incidence of myocardial infarction. An even longer-term follow-up of that study (15 years total) showed an 11% decrease in all-cause mortality among patients randomized to niacin. Because of its ability to reduce VLDL synthesis, niacin is also a first-line drug for treatment of hypertriglyceridemia.

Evidence is accumulating that xenoestrogens significantly affect gene expression in mammalian cells at doses far lower than their commonly considered toxic doses. Animal experiments suggest that in vivo effects of xenoestrogens follow non-monotonous dose response curves. However, whether such non-linearity is observed at the level of individual cells is unknown. To address this question, we developed a comprehensive database of dose-dependent transcriptomal effects of representative xenoestrogens in MCF-7 cells, which strongly express estrogen receptor a but not estrogen receptor β. For each xenoestrogen [estradiol, diethylstilbestrol, ethynylestradiol, Bisphenol A (BPA), genistein, daidzein, p-nonylphenol], transcriptomal snapshots were determined for 6 to 12 different doses in a single experiment using Affymetrix HG-U133-2+ microarray, and experiments were repeated at least five times. Databases were constructed to handle these ~480 snapshots (30~90 snapshots for each chemical). Exposure time was fixed to 48 hours, which was selected for best reproducibility of the snapshots. Exposure to correct doses was confirmed by qPCR determination of mRNA expression for WISP2 (estrogen-inducible marker) and BIK (estrogen-suppressible marker). The dose-specific snapshot data were integrated into the transcriptomal profile vectors for each chemical. Each element of the profile vector was a summary of multiple snapshots determined for a single concentration. The 50% effective doses (AC50) with 95% confidence intervals were calculated from the profile vectors for each of the 500 genes showing the best scores of curve fitting. By applying the B-spline curve fitting algorithm, mathematical models of the dose-dependent transcriptomal changes were produced form the profile vectors for each gene. These models generated estimations of transcriptomal profiles for unlimited numbers of concentrations within the concentrations ranges covered by the snapshot data. High-density virtual 3-D heatmaps drawn using these models revealed that mRNA expression of BPA-upregulated genes showed a small but significant peak at 10 pM~100 pM, once decreased at 1 nM~10 nM, and then increased again at 100 nM~10μM. This “second peak” of mRNA induction very low doses was specific to BPA as the virtual 3-D heatmaps of estradiol or other xenoestrogens did not show such peaks. In contrast, however, genes down-regulated by genistein showed significantly biphasic responses whereas genes up-regulated by the same ligand did not. Analyses on the AC50 distribution curves revealed that BPA-suppressible genes tend to be more sensitive than genes inducible by the same ligand with 2.3-fold differences in the average AC50, suggesting that relatively low concentrations of BPA may selectively suppress gene expression whereas its effects on gene induction are still weak. On the other hand, the AC50 distributions of estradiol-inducible and suppressible genes mostly overlapped, suggesting that the gene induction and suppression by this ligand occur simultaneously at all ranges of ligand concentrations. Thus, our comprehensive analyses on the dose-response characteristics of xenoestrogen-regulated gene expression show significant ligand species-dependent differences at low doses, suggesting that the low dose effects may occur even at the level of individual cells. This study was supported by Susan G. Komen for Cure grants FAS0703860 and KG090515 to TS.

I believe it is a valid assumption that most students applying to medical schools wish to become physicians because they look forward to caring for the sick with empathy and compassion and to be of benefit to mankind. In their altruism, they view their future role as healers who endeavor to provide comfort and support to those in need. Unfortunately, in the last three or four decades and perhaps even earlier, those goals and aspirations seem to diminish and even disappear as students traverse their medical education and training. And as they emerge as bona fide physicians and finish their residencies, it is often hard to detect signs of their original altruism.

Rare circulating tumor cells (CTCs) present in the bloodstream of patients with cancer provide a potentially accessible source for detection, characterization, and monitoring of nonhematological cancers. We previously demonstrated the effectiveness of a microfluidic device, the CTC-Chip, in capturing these epithelial cell adhesion molecule (EpCAM)-expressing cells using antibody-coated microposts. Here, we describe a high-throughput microfluidic mixing device, the herringbone-chip, or “HB-Chip,” which provides an enhanced platform for CTC isolation. The HB-Chip design applies passive mixing of blood cells through the generation of microvortices to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface. Efficient cell capture was validated using defined numbers of cancer cells spiked into control blood, and clinical utility was demonstrated in specimens from patients with prostate cancer. CTCs were detected in 14 of 15 (93%) patients with metastatic disease (median = 63 CTCs/mL, mean = 386 ± 238 CTCs/mL), and the tumor-specific TMPRSS2-ERG translocation was readily identified following RNA isolation and RT-PCR analysis. The use of transparent materials allowed for imaging of the captured CTCs using standard clinical histopathological stains, in addition to immunofluorescence-conjugated antibodies. In a subset of patient samples, the low shear design of the HB-Chip revealed microclusters of CTCs, previously unappreciated tumor cell aggregates that may contribute to the hematogenous dissemination of cancer.

Emergence of antiestrogen-resistant cells in MCF-7 cells during suppression of estrogen signaling is a widely accepted model of acquired breast cancer resistance to endocrine therapy. To obtain insight into the genomic basis of endocrine therapy resistance, we characterized MCF-7 monoclonal sublines that survived 21-day exposure to tamoxifen (T-series sublines) or fulvestrant (F-series sublines) and sublines unselected by drugs (U-series). All T/F-sublines were resistant to the cytocidal effects of both tamoxifen and fulvestrant. However, their responses to the cytostatic effects of fulvestrant varied greatly, and their remarkably diversified morphology showed no correlation with drug resistance. mRNA expression profiles of the U-sublines differed significantly from those of the T/F-sublines, whose transcriptomal responsiveness to fulvestrant was largely lost. A set of genes strongly expressed in the U-sublines successfully predicted metastasis-free survival of breast cancer patients. Most T/F-sublines shared highly homogeneous genomic DNA aberration patterns that were distinct from those of the U-sublines. Genomic DNA of the U-sublines harbored many aberrations that were not found in the T/F-sublines. These results suggest that the T/F-sublines are derived from a common monoclonal progenitor that lost transcriptomal responsiveness to antiestrogens as a consequence of genetic abnormalities many population doublings ago, not from the antiestrogen-sensitive cells in the same culture during the exposure to antiestrogens. Thus, the apparent acquisition of antiestrogen resistance by MCF-7 cells reflects selection of preexisting drug-resistant subpopulations without involving changes in individual cells. Our results suggest the importance of clonal selection in endocrine therapy resistance of breast cancer.

A biochemist's determination and brilliance unlocked the mystery of protein synthesis and created a new field of drug development.

Induction of mRNA for BIK proapoptotic protein by doxorubicin or gamma-irradiation requires the DNA-binding transcription factor activity of p53. In MCF7 cells, pure antiestrogen fulvestrant also induces BIK mRNA and apoptosis. Here, we provide evidence that, in contrast to doxorubicin or gamma-irradiation, fulvestrant induction of BIK mRNA is not a direct effect of the transcriptional activity of p53, although p53 is necessary for this induction. It is known that p53 up-regulated modulator of apoptosis (PUMA) mRNA is induced directly by the transcriptional activity of p53. Whereas gamma-irradiation induced both BIK and PUMA mRNA, only BIK mRNA was induced by fulvestrant. Whereas both fulvestrant and doxorubicin induced BIK mRNA, only doxorubicin enhanced the DNA-binding activity of p53 and induced PUMA mRNA. Small interfering RNA (siRNA) suppression of p53 expression as well as overexpression of dominant-negative p53 effectively inhibited the fulvestrant induction of BIK mRNA, protein, and apoptosis. Transcriptional activity of a 2-kb BIK promoter, which contained an incomplete p53-binding sequence, was not affected by fulvestrant when tested by reporter assay. Fulvestrant neither affected the stability of the BIK mRNA transcripts. Interestingly, other human breast cancer cells, such as ZR75-1, constitutively expressed BIK mRNA even without fulvestrant. In these cells, however, BIK protein seemed to be rapidly degraded by proteasome, and siRNA suppression of BIK in ZR75-1 cells inhibited apoptosis induced by MG132 proteasome inhibitor. These results suggest that expression of BIK in human breast cancer cells is regulated at the mRNA level by a mechanism involving a nontranscriptional activity of p53 and by proteasomal degradation of BIK protein.

To obtain insights into similarities and differences in the biological actions of related drugs or toxic agents, their transcriptomal signature profiles (TSPs) have been examined in a large number of studies. However, many such reports did not provide proper justification for the dosage criteria of each agent. Using a well characterized cell culture model of estrogen-dependent proliferation of MCF7 human breast cancer cells, we demonstrate how different approaches to dosage standardization exert critical influences on TSPs, leading to different and even conflicting conclusions. Using quantitative cellular response (QCR)-based dosage criteria, TSPs were determined by Affymetrix microarray when cells were proliferating at comparable rates in the presence of various estrogens. We observed that TSPs of the xenoestrogens (e.g., genistein or bisphenol A) were clearly different from the TSP of 17β-estradiol; namely, the former strongly enhanced expression of genes involved in mitochondrial oxidative phosphorylation, whereas the latter showed minimal effects. In contrast, TSPs for genistein and 17β-estradiol were indistinguishable by using the marker gene expression-based dosage criteria, conditions in which there was comparable expression of the mRNA transcripts for the estrogen-inducible WISP2 gene. Our findings indicate that determination and interpretation of TSPs in pharmacogenomic and toxicogenomic studies that examine the transcriptomal actions of related agents by microarray require a clear rationale for the dosage standardization method to be used. We suggest that future studies involving TSP analyses use quantitative and objective dosage standardization methods, such as those with quantitative cellular response or marker gene expression-based dosage criteria.